Interactions between proteins and/or their substrates or ligands are critical for normal cell function, physiologic signal transduction, as well as for therapeutic intervention in many pathophysiologic or disease-related processes. Proteins and peptides are capable of adopting compact, well-ordered conformations, and performing complex chemical operations, e.g., catalysis, highly selective recognition, etc. The three dimensional structure is the principal determinant that governs specificity in protein-protein and/or protein-substrate interactions. Thus, the conformation of peptides and proteins is central for their biological function, pharmaceutical efficacy, and their therapeutic preparation.
Protein folding is inextricably linked to function in both proteins and peptides because the creation of an “active site” requires proper positioning of reactive groups. Consequently, there has been a long-felt need to identify synthetic polymer or oligomers, which display discrete and predictable (i.e., stable) folding and oligomerizing propensities (hereinafter referred to as “foldamers”) to mimic natural biological systems. Insofar as these unnatural backbones are resistant to the action of proteases and peptidases, they are useful as probes having constrained conformational flexibility or as therapeutics with improved pharmacological properties, e.g., pharmacokinetic (PK) and/or pharmacodynamics (PD) features, such as potency and/or half-life. Whereas a naturally occurring polypeptide comprised entirely of α-amino acid residues will be readily degraded by any number of proteases and peptidases, foldamers, including chimeras of natural peptides and synthetic amino acid derivatives, mimetics or pseudopeptides, are not.
As noted above, the interest in foldamers stems in part from their resistance to enzymatic degradation. They are also interesting molecules because of their conformational behavior. The elucidation of foldamers having discrete conformational propensities akin to those of natural proteins has led to explorations of peptides constructed from β, γ-, or δ-amino acids. γ-Peptides containing residues bearing γ-substitution or α, γ-disubstitution or α, β, γ-trisubstitution have been shown to adopt a helical conformation defined by a 14-member turn that is stabilized by C═O(i)→NH(i+3) hydrogen bonds. Both the 314 and 2.512 helical backbones have been found suitable for the design of stabilized helical peptides useful for therapeutic purposes. For example, in order to cluster polar residues on one face of the helix, amphiphilic 314-helical β-peptides have been constructed from hydrophobic-cationic-hydrophobic- or hydrophobic-hydrophobic-cationic residue triads.
Considerable effort has been applied towards the development of artificial bio-inspired systems able to form predictable and homogeneous assemblies at the nanometer scale in aqueous conditions (Zhang, F., Nangreave, J., Liu, Y. & Yan, H. Structural DNA nanotechnology: state of the art and future perspective. J. Am. Chem. Soc. 136, 11198-11211 (2014); Lai, Y.-T., King, N. P. & Yeates, T. O. Principles for designing ordered protein assemblies. Trends Cell Biol. 22, 653-661 (2012); King, N. P. et al. Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science 336, 1171-1174 (2012); Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm cage designed by using protein oligomers. Science 336, 1129 (2012); Gradišar, H. & Jerala, R. Self-assembled bionanostructures: proteins following the lead of DNA nanostructures. J. Nanobiotechnology 12, 4 (2014); Lai, Y.-T. et al. Structure of a designed protein cage that self-assembles into a highly porous cube. Nat. Chem. 6, 1065-1071 (2014); King, N. P. et al. Accurate design of co-assembling multi-component protein nanomaterials. Nature 510, 103-108 (2014); Fletcher, J. M. et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595-599 (2013); Tebo, A. G. & Pecoraro, V. L. Artificial metalloenzymes derived from three-helix bundles. Curr. Opin. Chem. Biol. 25C, 65-70 (2015); Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362-366 (2013); and Bromley, E. H. C., Channon, K., Moutevelis, E. & Woolfson, D. N. Peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems. ACS Chem. Biol. 3, 38-50 (2008)). Although building units based on short peptides possess a number of advantages for nano-scale assembly—including synthetic availability, modularity and sequence diversity (Fletcher, J. M. et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595-599 (2013); Tebo, A. G. & Pecoraro, V. L. Artificial metalloenzymes derived from three-helix bundles. Curr. Opin. Chem. Biol. 25C, 65-70 (2015); Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362-366 (2013); and Bromley, E. H. C., Channon, K., Moutevelis, E. & Woolfson, D. N. Peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems. ACS Chem. Biol. 3, 38-50 (2008))—one limitation resides in the intimate connection between primary sequence and secondary structure. In this respect, non-natural synthetic oligomers designed to form predictable and well-defined secondary structures akin to those found in proteins—i.e. ‘foldamers’ (Gellman, S. H. Foldamers: A Manifesto. Acc. Chem. Res. 31, 173-180 (1998); Goodman, C. M., Choi, S., Shandler, S. & DeGrado, W. F. Foldamers as versatile frameworks for the design and evolution of function. Nat. Chem. Biol. 3, 252-262 (2007); and Guichard, G. & Huc, I. Synthetic foldamers. Chem. Commun. 47, 5933-5941 (2011))—obey different folding rules and thus provide new opportunities for creating self-assembled architectures with topologies similar to and beyond those of natural polypeptides. Additionally, in contrast to α-peptides, fully non-natural biopolymer backbones are likely—and in some cases proven—to be resistant to naturally occurring proteases (Johnson, L. M. & Gellman, S. H. α-Helix mimicry with α/β-peptides. Methods Enzymol. 523, 407-429 (2013); and Frackenpohl, J., Arvidsson, P. I., Schreiber, J. V. & Seebach, D. The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases. Chembiochem 2, 445-455 (2001)), a highly desirable characteristic for systems intended for bio-applications.
Advances in foldamer research have led to the discovery of a wide range of oligomeric backbones predisposed to adopt well-defined secondary structures, yet few reports have described the aqueous assembly of foldamers into homogeneous tertiary or quaternary arrangements—such examples being limited to the creation of de novo octameric helical bundles (Daniels, D. S., Petersson, E. J., Qiu, J. X. & Schepartz, A. High-resolution structure of a beta-peptide bundle. J. Am. Chem. Soc. 129, 1532-1533 (2007); and Wang, P. S. P., Nguyen, J. B. & Schepartz, A. Design and high-resolution structure of a β3-peptide bundle catalyst. J. Am. Chem. Soc. 136, 6810-6813 (2014)) and nanofibers (Pizzey, C. L. et al. Characterization of nanofibers formed by self-assembly of beta-peptide oligomers using small angle x-ray scattering. J. Chem. Phys. 129, 095103 (2008); and Pomerantz, W. C. et al. Nanofibers and lyotropic liquid crystals from a class of self-assembling beta-peptides. Angew. Chem. Int. Ed Engl. 47, 1241-1244 (2008)) formed from short amphiphilic helical β-peptides, and hybrid α/β-peptide tetrameric helical bundles obtained by reengineering the dimerization domain of the yeast GCN4 transcription factor (Home, W. S., Price, J. L., Keck, J. L. & Gellman, S. H. Helix bundle quaternary structure from alpha/beta-peptide foldamers. J. Am. Chem. Soc. 129, 4178-4180 (2007); and Giuliano, M. W., Horne, W. S. & Gellman, S. H. An alpha/beta-peptide helix bundle with a pure beta3-amino acid core and a distinctive quaternary structure. J. Am. Chem. Soc. 131, 9860-9861 (2009)).
Sequence remodeling of these backbones to yield well-defined yet dissimilar topologies has not been documented, except a rearrangement from parallel to anti-parallel helix topology in the α/β peptide bundle series (Giuliano, M. W., Home, W. S. & Gellman, S. H. An alpha/beta-peptide helix bundle with a pure beta3-amino acid core and a distinctive quaternary structure. J. Am. Chem. Soc. 131, 9860-9861 (2009)). Current aqueous foldamer quaternary assemblies are thus limited in terms of: 1) backbone diversity; 2) divergence from nature; 3) diversity of topology and, importantly; 4) control. An additional—more general—limitation is the paucity of high-resolution structural data available for aqueous foldamer quaternary assemblies (Daniels, D. S., Petersson, E. J., Qiu, J. X. & Schepartz, A. High-resolution structure of a beta-peptide bundle. J. Am. Chem. Soc. 129, 1532-1533 (2007); Wang, P. S. P., Nguyen, J. B. & Schepartz, A. Design and high-resolution structure of a β3-peptide bundle catalyst. J. Am. Chem. Soc. 136, 6810-6813 (2014); Horne, W. S., Price, J. L., Keck, J. L. & Gellman, S. H. Helix bundle quaternary structure from alpha/beta-peptide foldamers. J. Am. Chem. Soc. 129, 4178-4180 (2007); and Giuliano, M. W., Horne, W. S. & Gellman, S. H. An alpha/beta-peptide helix bundle with a pure beta3-amino acid core and a distinctive quaternary structure. J. Am. Chem. Soc. 131, 9860-9861 (2009))—and water-soluble foldamers in general. As such, there is a deficiency of pre-existing structures for use as templates for the design of new aqueous quaternary (and tertiary) assemblies.
Aliphatic oligoureas are a class of non-peptide α-helicomimetic foldamer which possess several features conducive for their use as self-organizing biomimetic building units. The chemical accessibility of the urea-based monomers permits the synthesis of urea oligomers bearing, yet not limited to, any of the 20 naturally occurring amino acid side-chains (Burgess, K., Shin, H. & Linthicum, D. S. Solid-Phase Syntheses of Unnatural Biopolymers Containing Repeating Urea Units. Angew. Chem. Int. Ed. Engl. 34, 907-909 (1995); and Douat-Casassus, C., Pulka, K., Claudon, P. & Guichard, G. Microwave-enhanced solid-phase synthesis of N,N′-linked aliphatic oligoureas and related hybrids. Org. Lett. 14, 3130-3133 (2012)). Importantly, the helicity of urea oligomers is largely unaffected by the nature of the side-chains used, making these foldamers highly robust and tunable (Fischer, L. et al. The canonical helix of urea oligomers at atomic resolution: insights into folding-induced axial organization. Angew. Chem. Int. Ed Engl. 49, 1067-1070 (2010); and Violette, A. et al. N,N′-linked oligoureas as foldamers: chain length requirements for helix formation in protic solvent investigated by circular dichroism, NMR spectroscopy, and molecular dynamics. J. Am. Chem. Soc. 127, 2156-2164 (2005)). In addition, oligoureas as short as 4-6 residues in length are able to adopt stable helical structures (Nelli, Y. R., Fischer, L., Collie, G. W., Kauffmann, B. & Guichard, G. Structural characterization of short hybrid urea/carbamate (U/C) foldamers: A case of partial helix unwinding. Biopolymers 100, 687-697 (2013)), bestowing an added dimension of versatility to these foldamers as self-assembling building blocks.
A key principle of foldamer research is to use biomolecules as inspiration for the design and development of molecules with functions and capabilities beyond those found in nature, such as catalysts or artificial bio-receptors with tailored ligand specificity. As function is intimately linked with structure, the creation of new and unique foldamer architectures is a necessary step towards the goal of developing foldamers with tailored/preternatural functions. However, the construction of novel foldamer structures can be challenging, particularly the creation of multi-component architectures, which require controlled, precise self-assembly. A seemingly even greater challenge than this is presented by the development of multimeric foldamer systems with the ability to self-assemble in aqueous conditions into precise, well-defined arrangements. The development of aqueous self-assembling foldamer systems is an important step towards the creation of foldamers with true bio-functions—such as bio-catalysis (Rufo, C. M., et al. Nat. Chem. 2014, 6 (4), 303; Wang, P. S. P., et al. J. Am. Chem. Soc. 2014, 136 (19), 6810; Tegoni, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (52), 21234; and Reig, A. J., et al. Nat. Chem. 2012, 4 (11), 900).
Until recently, examples of atomic-level structural elucidation of water-soluble foldamer quaternary assemblies were largely limited to β-amino acid containing backbones (Craig, C. J.; et al. ChemBioChem 2011, 12 (7), 1035; Giuliano, M. W., et al. J. Am. Chem. Soc. 2009, 131 (29), 9860; Home, W. S., et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (27), 9151; Horne, W. S. et al. J. Am. Chem. Soc. 2007, 129 (14), 4178; Daniels, D. S., et al. J. Am. Chem. Soc. 2007, 129 (6), 1532, Goodman, J. L., et al. J. Am. Chem. Soc. 2007, 129 (47), 14746). However, the strong propensity of short aliphatic oligourea foldamers to self-assemble in aqueous conditions into unique, precise quaternary arrangements, encompassing discrete helical bundles and extended tubular structures with water-filled pores was recently reported by the Applicant. Of particular note was the surprise discovery of an isolated hydrophobic cavity with a volume of around 500 Å3 within the helical bundle arrangements reported (FIG. 1). Although this volume is much smaller than that of nanocages built from the self-assembly of de novo designed proteins (Beck, T.; et al. Angew. Chem. Int. Ed. Engl. 2015, 54 (3), 937; Der, B. S.; et al. Nat. Biotechnol. 2013, 31 (9), 809; Lai, Y.-T., et al. Science 2012, 336 (6085), 1129; King, N. P., et al. Science 2012, 336 (6085), 1171.), it compares favourably with cavities engineered into peptide coiled-coils for small guest recognition (Ebalunode, J. O., et al. Bioorg. Med. Chem. 2009, 17 (14), 5133; Yadav, M. K., et al. Biochemistry 2005, 44 (28), 9723; Liu, J., et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (46), 16156; Johansson, J. S., et al. Biochemistry 1998, 37 (5), 1421). Cavities formed from water-soluble constructs represent a particularly enticing structural motif, as one could envisage the use of such a feature for, eventually, (drug) delivery, bio-sensing or catalysis. Over the last decade, supramolecular chemists have made remarkable progress towards the preparation of sophisticated organic and metal-organic containers—including covalent and self-assembled capsules—with a wide range of physicochemical properties (such as water solubility) and functions (such as dynamic systems able to adapt to the nature of the guest) (Rebilly, J.-N., et al. Chem. Soc. Rev. 2015, 44 (2), 467; Zarra, S., et al. Chem. Soc. Rev. 2015, 44 (2), 419; Biros, S. M., et al. Chem. Soc. Rev. 2007, 36 (1), 93; Ballester, P. Chem. Soc. Rev. 2010, 39 (10), 3810; and Conn, M. M., et al. Chem. Rev. 1997, 97 (5), 1647). Systems exploiting secondary structural folding have also been developed as capsules, and in particular, helical foldamers able to form internal cavities upon folding have emerged as a new and promising class of synthetic receptor (Singleton, M. L., et al. Angew. Chem. Int. Ed. Engl. 2014, 53 (48), 13140; Hua, Y., et al. J. Am. Chem. Soc. 2013, 135 (38), 14401; Wu, B. et al. Org. Lett. 2012, 14 (3), 684; Ferrand, Y., et al. J. Am. Chem. Soc. 2010, 132 (23), 7858; Juwarker, H., et al. Chem. Soc. Rev. 2009, 38 (12), 3316; Xu, Y.-X., et al. J. Org. Chem. 2009, 74 (19), 7267; Suk, J.-M., et al. J. Am. Chem. Soc. 2008, 130 (36), 11868; Garric, J., et al. Chemistry 2007, 13 (30), 8454; Garric, J., et al. Angew. Chem. Int. Ed. Engl. 2005, 44 (13), 1954; Inouye, M., et al. J. Am. Chem. Soc. 2004, 126 (7), 2022; and Tanatani, A., et al. Angew. Chem. Int. Ed. Engl. 2002, 41 (2), 325). Such capsule systems permit the optimisation of guest recognition to be achieved through rational sequence variation, allowing the volume and chemical nature of the cavity to be modified by the incorporation of appropriate residual building blocks. A recent example of such a fine tuning process describes the iterative design of helical foldamers with high selectivity for specific monosaccharides (Chandramouli, N., et al. Nat. Chem. 2015, 7 (4), 334). The vast majority of existing foldamer capsules, however, are restricted to monomeric helical foldamers with internal cavities, with few reports of foldamer capsules formed by the self-assembly of multi-molecular components (Singleton, M. L., et al. Angew. Chem. Int. Ed. Engl. 2014, 53 (48), 13140). Furthermore, foldamer capsule systems have not generally been employed and studied in aqueous environments (Suk, J.-M., et al. J. Am. Chem. Soc. 2008, 130 (36), 11868), thus there is a need for further development and diversification of foldameric capsule systems.
The design and construction of biomimetic self-assembling systems is a challenging yet potentially highly rewarding endeavour, contributing to the development of new biomaterials, catalysts, drug-delivery systems and tools for the manipulation of biological processes. While much has been achieved by engineering self-assembling DNA-, protein- and peptide-based building units, the design of entirely new, fully non-natural folded architectures resembling biopolymers (“foldamers”) with the ability to self-assemble into atomically precise nano-structures in aqueous conditions has proved exceptionally challenging. Furthermore, artificial synthetic molecules able to adopt well-defined stable secondary structures comparable to those found in nature (“foldamers”) have considerable potential for use in a range of applications such as bio-materials, bio-recognition, nano-machines and as therapeutic agents. The development of foldamers with the ability to bind and encapsulate “guest” molecules is of particular interest, as such an ability is a key step towards the development of artificial sensors, receptors and drug-delivery vectors. While significant progress has been reported within this context, foldamer capsules reported thus far are largely restricted to organic solvent systems. Therefore, there exists a need for fully non-natural folded architectures resembling biopolymers (“foldamers”) with the ability to self-assemble into atomically precise nano-structures in aqueous conditions, as well as a need for guest-encapsulation in aqueous conditions by a self-assembled foldameric capsule.